It has been estimated that two-thirds of Americans will experience at least one episode of back pain in their lifetime. Back pain affects more than half of people over the age of 65 and the treatment of back pain costs 50- 100 billion dollars per year in the United States. Most back pain is thought to result from damage to the intervertebral disc, which are located between each vertebra. Presently there is no cure for intervertebral discs that have been damaged either by physical injury or have degenerated. The goal of the parent R01 proposal is to determine the cellular and molecular mechanism(s) responsible for the formation of the intervertebral discs with a long-term goal to develop cell or protein-based therapies for disc degeneration. The Harfe laboratory has extensive expertise in developmental biology and genetics, which allowed us to quickly identify the embryonic notochord as the cell population that forms the entire middle part of the mature disc called the nucleus pulposus (this was one of the aims of the parent grant and this work has now been published). It is this region of the disc that is usually damaged in patients that have severe back pain. In this proposal, we describe pilot experiments to develop a cell-based therapy to heal damaged or degrading discs using purified notochord cells and a model of disc degeneration. This project is a new collaboration between the Harfe laboratory at the University of Florida and the Elliott laboratory at the University of Pennsylvania. The Elliott laboratory has extensive expertise in disc mechanical function and degeneration, animal models of disc degeneration, and disc tissue engineering. Together, the two groups are uniquely equipped to translate our understanding of nucleus pulposus development into cell-based therapies for disc degeneration. The therapeutic opportunity of using cells that normally form the nucleus pulposus to heal damaged discs was identified as a future goal, beyond the 5-year scope, of the parent grant. Due to our recent identification of nucleus pulposus precursor cells it is now possible to begin experiments to directly test possible therapeutic roles for these cells. The interdisciplinary partnership with Dr. Elliott significantly enhances the scope of the parent grant by immediately translating the basic understanding of the developmental mechanism responsible for disc formation discovered by Dr. Harfe to treating disc degeneration. In this proposal we will begin to test the therapeutic potential of notochord cells by first injecting purified notochord cells into the adult nucleus pulposus of normal mice (Aim 1). Our recent exciting discovery that all cells present in the mature mouse nucleus pulposus are derived from the embryonic notochord suggests that notochord cells may be able to function as disc stem cells. To test if purified notochord cells can differentiate into all cell types found in the mature nucleus pulposus we propose to inject these cells, marked with LacZ, into the adult mouse nucleus pulposus. Their ability to survive, proliferates, and form all nucleus pulposus cell types will be determined using molecular markers.